How to design primer for sequencing unknown DNA - (Mar/28/2006 )

Hello,

I have an question about primer design for sequencing.
If you want to design a primer, it is nessesary to have de sequence of your gene.

But what if you dont have the sequence of your ampflicatet gene?
I red that it is possible to paste your gene it into a vector and design primers near the MCS.
But how can you ampflicate your gene if you dont have primers because the sequence of your gene is unknow?

Maybe a stuped question, but I cant find anathing on internet...

Roy

Yes you do need the sequence. It is possible to clone the frag into an MCS and then prime the plasmid sequences but you need to know which restriction enzymes are going to be apporopriate which means knowing the sequence.

There is an alternative. Radiolabel you fragment with T4 kinase and then use Maxam-Gilbert techniques to cleave at G and A bases. There are protocol easily available that allow you to cleave selectively at G, A and C residues. The T bases fall out by elimination (not in a chemical sense).

Resolve the cleaved products on a high voltage polyacrylamide gel. It's quite sensitive and you might just have enough DNA. How much do you have?

Thank you for your help! iam making a poster presentation about primer design so it is not for a experiment.

Do you know some very good websites where they explain a lot about primer design?
I have found some good websites with google. Do you know some more good websites?

Hi Roy,

what species are you working on?

What I do is make an alignment of the gene I am interested in from other species, then design the primers from the consensus of the other sequences.

It works quite well.

try a vector like pGemTeasy - T/A cloning (ie. ligate your PCR product straight into the vector, assuming your Taq tails the ends of the product), and use primers designed to the polymerases in the vector - T7 and SP6 for example...